Many pathogens grow within host cells by building membrane-bound replication compartments essential for growth. The compartments provide important protection from host innate immune cytoplasmic surveillance systems and interfere with trafficking organisms into degradative compartments. Growth in the compartment is driven by microbial proteins which promote compartment construction, protection against innate immune surveillance and provide regulatory control over the host cell. How these different arms of the pathogen are coordinated is poorly understood. A bacterium that uses this strategy is Legionella pneumophila, which grows in a vacuole within macrophages during pneumonic disease. A group of over 300 Legionella proteins are translocated into host cells, controlling all aspects of the intracellular lifestyle, including formation of the replication compartment, blocking host cell translation, and preventing cytosolic recognition of the replication compartment. The organism hijacks host tubular endoplasmic reticulum (ER) as one of the earliest steps in replication compartment formation, an event promote by the bacterial Sde proteins. The proposed studies will test two models for Sde function. First, it will test the model that the Sde proteins controls ER tubule formation, replication compartment formation, and immune avoidance by promoting a three-step pathway. Secondly, it will test the model that manipulation of host translation initiation plays a role in coordinating replication vacuole construction and immune avoidance. The first model proposes that the Sde deubiquitinase activity liberates free ubiquitin (Ub) from the polyUb on the replication compartment. This would serve as a pool for ADPribosyltransferase and phosphodiesterase domains to promote Ub modification of the tubular ER protein Rtn4 and host translation initiation factors (eIFs). In the final step, the protein family masks the replication compartment with a phosphoribose to prevent recognition by the host autophagy pathway. The model will be tested by manipulating polyUb pools in the cell, performing electron microscopy on mutants deranged in this pathway, and reconstructing tubular ER rearrangements in a cell- free system. To determine the role that targeting of eIF proteins plays in these processes, the targets will be verified using purified components and the regulated initiation step will be identified. In addition, it will be determined if eIF proteins are inactivated to remove ribosomes from rough ER and allow close docking of ER sheets to the replication compartment, and if eIF manipulation skews the host translational response to favor bacterial replication. Extensive preliminary studies support the proposed experiments, providing a solid underpinning for this work. Throughout these studies, the objective is to identify a weak link in the microbial strategy of manipulating ER tubules to promote intracellular growth, with an eye toward developing antimicrobials that target this process.